Heparin-induced thrombocytopenia (HIT), the most frequent drug-induced immune-mediated type of thrombocytopenia (low platelet level in the blood), is an important and sometimes life-threatening complication of heparin therapy. Heparin is the most widely used intravenous anti-coagulant and one of the most widely prescribed drugs in the U.S. More than one trillion units are administered annually to approximately 12 million patients. Intravenous heparin is commonly used for the prophylaxis and treatment of thromboembolic disease, as well as numerous other applications including certain types of heart and lung disorders, and during or after a variety of surgery including open heart, bypass, dialysis and orthopedic procedures. Heparin is also used for diagnostic and therapeutic interventional radiologic procedures. HIT can be triggered by standard therapeutic dose heparin, low-dose (prophylactic treatment), low molecular weight heparin, unfractionated heparin and even by minute quantities given to flush intravascular catheters.
HIT is classified into Type I and Type II, Type I being benign and Type II severe. Type I occurs early after heparin initiation, when the platelet levels are reduced only slightly and usually return to normal even if heparin treatment is continued. Thromboembolic complications are rare, and Type I HIT is not antibody-mediated.
In contrast, Type II HIT is a clinicopathologic syndrome caused by platelet activating antibodies that recognize complexes of platelet factor 4 (PF4) and heparin (PF4/heparin) (Amiral et al., Thromb. Haemost. 1992, 68:95-96). Thrombocytopenia occurring within 5-14 days of administration of heparin is the most common clinical effect. The most severe complication of HIT is the occurrence of venous and arterial thrombosis (HIT-associated thrombosis or HITT). Venous thrombosis can result in limb gangrene and the need for limb amputation. Other symptoms of Type II HIT may include cutaneous reactions, from a simple allergic reaction to lesions and necrosis.
The basic pathogenesis of HIT is reasonably well characterized. Upon exposure to exogenous heparin, multi-molecular complexes composed of PF4 and heparin form. The binding of PF4 to heparin in these complexes is associated with a conformational change in PF4, exposing a neoepitope (Reilly, Semin. Dial. 2003, 16:54-60). The present inventors previously reported that many polyanionic compounds other than heparin (e.g., polyanions such as polyvinyl sulfonate) can form complexes with PF4 and cause similar conformation change in the molecule (Visentin et al., J. Lab. Clin. Med. 2001, 138:22-31; U.S. Pat. No. 5,972,718, incorporated herein by reference). This heparin-dependent neoepitope elicits an immune response in some patients; the antibody response is usually an IgG type of antibody, although IgA and IgM types have also been implicated (Amiral et al., Br. J. Haematol. 1996, 92:954-59). The antibody binds to the PF4/heparin complex, creating an immune complex that binds to immunoglobulin receptors on the platelet surface (FcγRIIA or CD32). Aggregation of FcγRIIA receptors by the immune complex triggers platelet activation and aggregation through transmembrane signaling. In addition, during the process of platelet activation, platelets release phospholipid microparticles derived from the cell membrane. These microparticles support the enzymatic reactions of the coagulation cascade, leading to thrombin formation; consequently, the release of these microparticles into the circulation is thrombogenic. Further, the PF4/heparin immune complexes can bind to glycosaminoglycans on the endothelium, leading to endothelial injury. The endothelial damage may also participate in the “thrombotic storm” associated with this syndrome.
Early diagnosis of HIT is essential to reduce morbidity and mortality. The diagnosis of HIT is usually based on clinical abnormalities including thrombocytopenia with or without thrombosis and the detection of antibodies to the PF4/heparin complex (termed PF4/heparin antibodies). There are two major types of assays for the detection of heparin dependant antibodies: functional assays and PF4-dependent antigen immunoassays. Functional assays include the serotonin release assay (SRA; Sheridan et al., Blood 1986, 67:27-30), the platelet aggregation assay (Chong et al., Thromb. Haemost. 1993, 69:344-50) and the heparin-induced platelet activation (HIPA) assay (Greinacher et al., Thromb. Haemost. 1991, 66:734-36). Functional assays are technically difficult to perform and are considered to be complex specialty assays.
Enzyme linked immunoassays (ELISAs) are the most frequently used PF4-dependent antigen immunoassay (Greinacher et al., Hematol. 1994, 34:381-85; Visentin et al., J. Clin. Invest. 1994, 93:81-88; see also U.S. Pat. Nos. 5,466,582, 5,972,717, 5,972,718, 6,964,854, 7,011,953 and 7,728,115; U.S. Patent Application Pub. No. 2010/0015647; and Int'l Pub. No. WO 2010/024271, all of which are incorporated herein by reference). Commercially available ELISAs for the detection of PF4/heparin antibodies include PF4 IgG™ and PF4 Enhanced® (both from Gen-Probe GTI Diagnostics, Waukesha, Wis.); Asserachrom® HPIA and Asserachrom® HPIA-IgG (both from Diagnostica Stago, Asnières, France); Technozym® HIT IgG (Technoclone, Vienna, Austria); and Zymutest HIA IgGAM (Hyphen Biomed, Neuville, France). ELISAs, while not particularly complex, are generally not available on a point-of-care basis.
Although HIT is a true clinicopathologic syndrome, its diagnosis still rests primarily on clinical grounds since laboratory tests may not be available locally or may not be available in a sufficiently timely manner. Owing to the high risk of HIT-associated thrombosis, antithrombotic therapy with alternative anticoagulants should be started immediately when serologic assays confirm clinical suspicion. Readily available results, obtained from immunoassays for rapid detection of PF4/heparin antibodies, may be combined with the pretest estimation of clinical probability in order to exclude and/or confirm the diagnosis of HIT, thereby assisting physicians in the time-sensitive clinical management of HIT.
A number of rapid PF4-dependent antigen immunoassays have been described, e.g., the particle immunofiltration assay (PIFA) disclosed in U.S. Patent Application Pub. No. 2006/0172438 and corresponding Int'l Pub. No. WO 2006/042089, as well as the lateral flow immunoassay (LFIA) disclosed in and U.S. Patent Application Pub. No. 2010/0255510 and corresponding Int'l Pub. No. WO 2009/111254, all incorporated herein by reference. Several rapid PF4-based immunoassays have been developed commercially. ID-PaGIA Heparin/PF4 Antibody Test (DiaMed/Bio-Rad, Cressier, Switzerland) is a rapid particle gel immunoassay that utilizes PF4 coated onto synthetic polymer particles. PIFA® Heparin/PF4 Rapid Assay (Akers Biosciences, Thorofare, N.J.) is a particle immunofiltration assay that uses dyed microparticles coated with purified PF4 protein. Enhancing agents promote rapid matrix formation of these microparticles in the presence of PF4/heparin antibodies, and visual color results are produced through the interaction of these microparticles with a membrane-filtration system. Finally, Milena® QuickLine HIT (Milenia Biotec, Gieβen, Germany) is a rapid lateral flow immunoassay wherein goat anti-human IgG antibodies immobilized on the nitrocellulose membrane bind human IgG antibodies previously captured by a PF4/polyanion complex which is detected using intensely colored gold nanoparticles.
Despite the commercial availability of various PF4-dependent antigen immunoassays, there remains a significant need for simple, rapid, sensitive and specific methods for detecting PF4/heparin antibodies in fluid samples, particularly ones that can be performed in the point-of-care settings without relying on sophisticated laboratory equipment. The present invention addresses this need.